Peptide-based delivery agent and method of making and using the same

Abstract

Disclosed herein is a delivery agent that facilitates effective delivery of drugs and other compounds across lipid layers. The delivery agent disclosed herein provides lipid solubility under selected conditions and aqueous solubility under other conditions, and can effectively deliver compounds into the cell cytosol. The disclosed delivery agent also avoids deleterious interactions with serum and thus provide efficient in vivo delivery of therapeutic agents.

1 . A peptide-based delivery agent comprising:
 a lytic peptide group having a structure according to a formula [X 1 Y 1 Y 1 X 1 ] m , wherein
 each X 1  independently for each occurrence is an amino acid, or a derivative thereof, provided that at least one X 1  of the lytic peptide group is a basic amino acid or an acidic amino acid; 
 each Y 1  independently for each occurrence is a non-polar amino acid or a derivative thereof; and 
 m is an integer selected from 2 to 8; 
   a cleavable linker group;   a mask peptide group having a structure according to a formula [X 2 Y 2 Y 2 X 2 ] m′ , wherein
 each X 2  independently for each occurrence is an amino acid, or a derivative thereof, provided that at least one X 2  of the mask peptide group is glutamine; 
 each Y 2  independently for each occurrence is a non-polar amino acid or a derivative thereof; and 
 m′ is an integer selected from 2 to 8. 
   
     
     
         2 . The peptide-based delivery agent of  claim 1 , further comprising:
 (i) an anchor group selected from a heteroaliphatic group, a dibenzocyclooctyne compound, an antibody or antibody fragment, a biotin group, an avidin group, a streptavidin group, or a neutravidin group; a targeting group selected from a cell, an antibody or antibody fragment, a peptide, a biomimetic peptide, an aptamer, a sugar, or a small targeting molecule; or a combination thereof;   (ii) an N-terminus group; and   (iii) a C-terminus group;   
       wherein the peptide-based delivery agent has a structure according to Formula IA or Formula IB 
       
         
           
           
               
               
           
         
       
       wherein
 the cleavable linker is an amino acid sequence that is two to ten amino acids in length; 
 the anchor group, if present, is selected from a heteroaliphatic group, a dibenzocyclooctyne compound, an antibody or antibody fragment, a biotin group, an avidin group, a streptavidin group, or a neutravidin group; 
 the targeting group, if present, is selected from a cell, an antibody or antibody fragment, a peptide, a biomimetic peptide, an aptamer, a sugar, or a small targeting molecule; or a combination thereof; 
 the C-terminus group comprises an amine-terminated glycine moiety; and 
 the N-terminus group comprises a capping group or a fluorophore. 
 
     
     
         3 . The peptide-based delivery agent of  claim 1 , wherein each X 1  independently for each occurrence is glutamic acid, glutamine, arginine, alanine, aspartic acid, or a derivative thereof, provided that if an X 1  is alanine, then at least one further X 1  is glutamine, glutamic acid, aspartic acid, arginine, or a derivative thereof. 
     
     
         4 . The peptide-based delivery agent of  claim 1 , wherein each Y 1  independently for each occurrence and each Y 2  independently for each occurrence is leucine, α-methyl leucine, alanine, or a derivative thereof. 
     
     
         5 . The peptide-based delivery agent of  claim 1 , wherein each X 2 , other than the at least one X 2  of the mask peptide group that is glutamine as recited by  claim 1 , independently for each occurrence is glutamine, glutamic acid, aspartic acid, alanine, or a derivative thereof. 
     
     
         6 . The peptide-based delivery agent of  claim 1 , wherein m is 3 and m′ is 3; or m is 4 and m′ is 4. 
     
     
         7 . The peptide-based delivery agent of  claim 2 , wherein each X 1  is glutamic acid; each Y 1  and each Y 2  is leucine; each X 2 , other than the at least one X 2  of the mask peptide group that is glutamine, is glutamine or alanine; m is 3; and m′ is 3; or each X 1 , other than the X 1  that is directly attached to the cleavable linker, is aspartic acid; each Y 1  and each Y 2  is leucine; each X 2 , other than the at least one X 2  of the mask peptide group that is glutamine, is glutamine or alanine; m is 3; and m′ is 3. 
     
     
         8 . The peptide-based delivery agent of  claim 1 , wherein the cleavable linker is cleavable by cathepsin B and comprises an amino acid sequence two to ten amino acids in length. 
     
     
         9 . The peptide-based delivery agent of  claim 1 , wherein the cleavable linker comprises an amino acid sequence at least 70% identical or 85% identical to SEQ ID NO: 3. 
     
     
         10 . The peptide-based delivery agent of  claim 2 , wherein the anchor group comprises:
 (i) an amino acid portion that is a lysine moiety; and   (ii) a tail group that comprises (a) a functional group provided by a 2-aminoethyl hydrogen carbonate group, a 4-aminobutanoic acid group, a —CH(NH 2 )C(O)—group, or a —CH(N + Me 3 )C(O)— group; and (b) an aliphatic tail bound to the functional group comprising 6 to 12 carbon atoms.   
     
     
         11 . The peptide-based delivery agent of  claim 10 , wherein the anchor group is selected from 
       
         
           
           
               
               
           
         
       
     
     
         12 . The peptide-based delivery agent of  claim 2 , wherein a plurality of anchor groups is present. 
     
     
         13 . The peptide-based delivery agent of  claim 2 , wherein the lytic peptide group provides an N-terminus group of the peptide-based delivery agent and the anchor group provides a C-terminus group of the peptide-based delivery agent, wherein (i) the N-terminus group is bound to a carbonyl-containing group; and (ii) the C-terminus group is bound to an amine-terminated glycine moiety, a fluorophore, or a combination thereof. 
     
     
         14 . The peptide-based delivery agent of  claim 2 , wherein:
 (i) the lytic peptide group has a structure [ELLE] 3 ; the cleavable linker has an amino acid sequence of SEQ ID NO: 3; the mask peptide group has a structure [QLLQ] 3 ; and the peptide-based delivery agent comprises the anchor group; and wherein (a) the lytic peptide group provides an N-terminus that is bound to an acetyl group, and (b) the anchor group is bound to an amine-terminated glycine moiety having a structure -G-Nal-G′, wherein G′ is a modified glycine comprising a —C(O)—N(R a ) 2  group, wherein each R a  independently is hydrogen or aliphatic; or   (ii) the lytic peptide group has a structure [ELLE] 4 ; the cleavable linker has an amino acid sequence of SEQ ID NO: 3; the mask peptide group has a structure [QLLQ] 4 ; and peptide-based delivery agent comprises two anchor groups and are bound directly to one another; and wherein (a) the lytic peptide group provides an N-terminus that is bound to an acetyl group, and (b) the anchor group is bound to a modified glycine comprising a —C(O)—N(R a ) 2  group, wherein each R a  independently is hydrogen or aliphatic; or   (iii) the lytic peptide group has a structure [QLLE]-[QLLQ]-[QLLE]; the cleavable linker has an amino acid sequence of SEQ ID NO: 3; the mask peptide group has a structure [QLLE]-[QLLQ]-[QLLE]; and the peptide-based delivery agent comprises the anchor group; and wherein (a) the lytic peptide group provides an N-terminus that is bound to an acetyl group, and (b) the anchor group is bound to an amine-terminated glycine moiety having a structure-G-K-G′, wherein G is glycine, K is lysine, and G′ is a modified glycine comprising a —C(O)—N(R a ) 2  group, wherein each R a  independently is hydrogen or aliphatic;   (iv) the lytic peptide group has a structure [ELLE] 3 ; the cleavable linker has an amino acid sequence of SEQ ID NO: 3; the mask peptide group has a structure [QLLA]-[QLLA]-[QLLQ]; and the peptide-based delivery agent comprises the anchor group; and wherein (a) the lytic peptide group provides an N-terminus that is bound to an acetyl group, and (b) the anchor group is bound to an amine-terminated glycine moiety having a structure -G-K-G′, wherein G is glycine, K is lysine, and G′ is a modified glycine comprising a —C(O)—N(R a ) 2  group, wherein each R a  independently is hydrogen or aliphatic; or   (v) the lytic peptide group has a structure [DLLD]-[DLLD]-[DLLE]; the cleavable linker has an amino acid sequence of SEQ ID NO: 3; the mask peptide group has a structure [QLLQ] 3 ; and the peptide-based delivery agent comprises the anchor group; and wherein (a) the lytic peptide group provides an N-terminus that is bound to an acetyl group, and (b) the anchor group is bound to an amine-terminated glycine moiety having a structure-G-K-G′, wherein G is glycine, K is lysine, and G′ is a modified glycine comprising a —C(O)—N(R a ) 2  group, wherein each R a  independently is hydrogen or aliphatic.   
     
     
         15 . The peptide based delivery agent of  claim 2 , having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 12, 272, 273, or 274 and wherein K at position 32 comprises a linking group selected from Formula A, B, or C, and wherein the linking group is attached to one or more DBCO groups. 
     
     
         16 . A composition, comprising:
 the peptide-based delivery agent of  claim 1 ; and   a therapeutic agent selected from a chemotherapeutic, an antisense molecule, a therapeutic antibody, an immune therapeutic, an antibiotic, an antidepressant, or a combination thereof.   
     
     
         17 . The composition of  claim 16 , wherein the therapeutic agent is a morpholino comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 229, 240, 241, 242, 243, and 244. 
     
     
         18 . The composition of  claim 16 , wherein (i) the peptide-based delivery agent comprises an antibody as the anchor group or the targeting group (ii) the therapeutic agent is a morpholino and wherein the morpholino is indirectly covalently bound to the peptide-based delivery agent through a linking group. 
     
     
         19 . The composition of  claim 16 , formulated for administration by injection, aerosol delivery, intranasal administration, oral administration, topical administration, or a combination thereof. 
     
     
         20 . A method, comprising contacting a cell in vitro or in vivo with the composition of  claim 16 , wherein the peptide-based delivery agent of the composition delivers the therapeutic agent to the cell's cytosol and contacting the cell with the composition induces:
 lysis of a membrane following internalization of the therapeutic agent and the peptide-based delivery agent into the cell; or   pore formation in a membrane following internalization of the therapeutic agent and the peptide-based delivery agent into the cell; or   local disruption/destabilization of a membrane following internalization of the therapeutic agent and the peptide-based delivery agent into the cell.   
     
     
         21 - 23 . (canceled) 
     
     
         24 . A method of identifying a therapeutic compound, comprising:
 contacting a cell with the peptide-based delivery agent of  claim 1  and one or more compounds;   determining an effect of the one or more compounds on the contacted cell; and   comparing the effect of the one or more compounds on the contacted cell to a control; wherein a differential effect of the one or more compounds on the contacted cell relative to the control indicates that the one or more compounds is a therapeutic compound.   
     
     
         25 . The method of  claim 24 , wherein the method is a quantitative high-throughput screening method and wherein the effect of the one or more compounds on the contacted cell comprises:
 reduced survival of the contacted cell compared to the control;   increased survival of the contacted cell compared to the control;   induction of a phenotype of interest in the contacted cell compared to the control;   increased expression of one or more genes in the contacted cell compared to the control; and/or   decreased expression of one or more genes in the contacted cell compared to the control.   
     
     
         26 - 27 . (canceled)